DNA fingerprinting (also known as DNA profiling) using short tandem repeats (STRs) has become the method of choice for human identification in forensic sciences, finding applications in different circumstances such as determination of perpetrators of violent crime, resolving unestablished paternity, and identifying remains of missing persons or victims of mass disaster. STRs are highly polymorphic microsatellite regions of 2–7 bp localized in noncoding regions of DNA. Every individual has a different pattern of STRs due to a different number of repeats and micro-variation in the sequences of the repeats.
The FBI and the forensic science community typically use 13 separate STR loci (the core CODIS loci) in routine forensic analysis. (CODIS refers to the Combined DNA Index System that was established by the FBI in 1998). If two DNA samples have identical lengths at all 13 loci, the probability that the two samples did not originate from the same individual is approximately one to ten billion. The courts generally accept this identification as definitive evidence that the individuals in question are the same. It is believed that STR analysis will remain the technique of choice in forensic science for DNA fingerprinting for the next decade, and that the number of loci used in this analysis will perhaps increase from 13 to 20.
Generally, to perform a DNA fingerprinting experiment based on STR analysis, the regions of DNA corresponding to each of the 13 STR loci are excised from sample DNA using appropriate restriction enzymes. The regions are then amplified using PCR and labeled with a dye or fluorescent molecule. The length of the DNA molecules is then determined using polyacrylamide gel electrophoresis (PAGE) or other known electrophoretic separation techniques, see, e.g., John M. Butler “Forensic DNA Typing” Academic Press, 2001.
Electrophoresis is a separation technique based on size, i.e., shorter DNA molecules migrate more rapidly down a gel or capillary than longer DNA molecules. The population of molecules (in this case, STR regions) is thus separated by size (or repeat length), and the final position of the DNA is determined by visualizing the staining pattern of the dye or fluorescent molecule. While there are miniature systems with an array of electrophoretic columns for this measurement, the number of STR regions and samples that can be identified using these miniature systems is relatively small.
Although still in their infancy, several DNA fingerprinting methods using microarrays have been proposed. For example, R. Radtkey et al., in “Rapid, high fidelity analysis of simple sequence repeats on an electronically active DNA chip” Nucleic Acids Research, 28:E17 (2000), offer a high stringency approach for discriminating STR alleles based on active microarray hybridization. A sandwich hybrid is assembled, in which proper base stacking of juxtaposed terminal nucleotides results in a thermodynamically favored complex. The increased stability of this complex relative to non-stacked termini and/or base pair mismatches is used to determine the identification of STR alleles. While this method has the advantage of being able to test many samples and STRs in a small instrument, it has the disadvantage of requiring the use of a special electronically active DNA array to allow discrimination of subtle hybridization differences between repeats of similar lengths. Thus, this method has not been widely adopted.
Another proposed microarray method involves the use of ligase and/or polymerase to detect the length of a VNTR (variable number of tandem repeats). For example. U.S. Pat. No. 6,150,095 discloses a technique in which the length of a VNTR is detected by hybridizing a target to a short probe to form a duplex, incubating the duplex with labeled nucleotides, and monitoring chain extension of the probe as an indication of the length of the variable number repeat section of the target. Other methods to determine the length of VNTR involve the use of ligation of tags combined with base extension. VNTR-based DNA fingerprinting has largely been superseded by STR-based DNA fingerprinting.
U.S. Pat. No. 5,753,439 discloses a method of using nuclease to nick mismatched base pairs followed by nick translation using DNA polymerase. With this method, target DNA is labeled and hybridized to a differently labeled probe. Mismatched bases due to differences in the length of the repeat region between the probe and the target are nicked with nuclease, and the remainder of the probe or target is elongated using nick translation, thereby displacing the label on the target or probe. This method is complicated and thus has not gained wide adoption.
Accordingly, there is a need in the art to develop new, simple DNA fingerprinting methods utilizing widely available microarrays for rapid determination of individual identity.